Expression of soluble, glycosylated and correctly folded dengue virus NS1 protein in Pichia pastoris.
Protein Expr Purif
; 162: 9-17, 2019 10.
Article
en En
| MEDLINE
| ID: mdl-31077812
ABSTRACT
The dengue virus (DENV) non structural protein 1 (NS1) is a 46-55â¯kDa protein that exists as homodimer inside cells and as hexamer in the extracellular milieu. Several lines of evidence have demonstrated that the biochemical and structural properties of recombinant NS1 (rNS1) vary depending on the protein expression system used. Aiming to improve current tools for studying NS1 multiple roles, a recombinant tag-free NS1 protein was expressed and purified from P. pastoris yeast cells. The best expression condition was achieved using GS115 strain and induction for 72â¯h with 0.7% methanol addition every 24â¯h. Total secreted rNS1 reached 2.18â¯mg in 1â¯L culture and the final yield of the purified protein was 1â¯mg per liter of culture (recovery yield of approximately 45.9%). Size-exclusion chromatography and treatment with EndoH and PNGase indicate that it exists as an N-glycosylated homodimer. Moreover, an ELISA assay with specific DENV anti-NS1 antibody that recognizes conformational epitopes revealed that rNS1 has most of its conformational epitopes preserved. The expression of rNS1 in its native conformation is an important tool for further studies of its role in DENV pathogenesis and replication.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
3_ND
Problema de salud:
3_dengue
/
3_neglected_diseases
Asunto principal:
Pichia
/
Proteínas no Estructurales Virales
/
Virus del Dengue
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2019
Tipo del documento:
Article
País de afiliación:
Brasil